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Comparative Mapping of Disease Resistance Genes in Lathyrus Spp

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Comparative Mapping of Disease Resistance Genes in Lathyrus Spp Comparative mapping of disease resistance genes in Lathyrus spp. using model legume genetic information Nuno Felipe Alves de Almeida Dissertation presented to obtain the Ph.D degree in Biology Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa Oeiras, July, 2015 PhD Supervisors Maria Carlota Vaz Patto, PhD Diego Rubiales, PhD The work presented in this thesis was performed mainly at: Under the supervision of Maria Carlota Vaz Patto, PhD Under the supervision of Diego Rubiales, PhD The student, Nuno Felipe Alves de Almeida received financial support from Fundação para a Ciência e Tecnologia and Fundo Social Europeu in the scope of Quadro Comunitário de Apoio through the PhD Fellowship SFRH/BD/44357/2008. iii ISBN: 978-989-20-5841-2 Table of Contents LIST OF FIGURES .............................................................................. XIII LIST OF TABLES ................................................................................ XV LIST OF ADDITIONAL FILES .............................................................. XVII ACKNOWLEDGMENTS / AGRADECIMENTOS ......................................... XIX RESUMO .......................................................................................... XXI ABSTRACT ...................................................................................... XXV LIST OF ABBREVIATIONS ................................................................. XXIX CHAPTER 1 - GENERAL INTRODUCTION ................................................. 1 1.1. Introduction ........................................................................ 3 1.2. Lathyrus sativus and L. cicera: Origin and systematic ... 4 1.3. Lathyrus sativus varietal groups ...................................... 6 1.4. Genetic resources and utilization ..................................... 7 1.5. Major breeding achievements ........................................... 9 1.6. Specific goals in current breeding .................................. 10 1.7. Biotic stresses.................................................................. 11 1.7.1. Rust ........................................................................ 12 1.7.2. Powdery mildew ...................................................... 13 1.7.3. Ascochyta blight ...................................................... 13 1.8. Breeding methods and specific techniques ................... 15 1.9. Integration of new biotechnologies in breeding programmes ..................................................................... 17 v Table of Contents ___________________________________________________________________ 1.10. Objectives ......................................................................... 20 1.11. Acknowledgments ........................................................... 21 1.12. References ........................................................................ 21 CHAPTER 2 - TRANSFERABILITY OF MOLECULAR MARKERS FROM MAJOR LEGUMES TO LATHYRUS SPP. FOR THEIR APPLICATION IN MAPPING AND DIVERSITY STUDIES ......................................... 31 2.1. Abstract ............................................................................ 33 2.2. Introduction ...................................................................... 34 2.3. Methods ............................................................................ 36 2.3.1. Plant Material and DNA isolation ................................... 36 2.3.2. Microsatellite markers .................................................... 38 2.3.3. Intron-Targeted Amplified Polymorphic markers, Defence-Related Genes and Resistance Gene Analogues ..................................................................... 39 2.3.4. Sequencing ................................................................... 40 2.3.5. Segregation and diversity studies .................................. 40 2.3.6. Cleaved Amplified Polymorphic Sequence and derived Cleaved Amplified Polymorphic Sequence markers for mapping ......................................................................... 41 2.4. Results and discussion ................................................... 42 2.4.1. Transferability of microsatellites to Lathyrus spp. ........... 44 2.4.2. Transferability of Intron-Targeted Amplified Polymorphic markers, Defence-Related and Resistance Gene Analogues to Lathyrus spp. ........................................... 50 2.4.3. Usefulness of cross amplified markers on Lathyrus spp. linkage mapping ............................................................ 52 vi Table of Contents ___________________________________________________________________ 2.4.4. Usefulness of cross amplified markers on Lathyrus spp. diversity studies ............................................................. 54 2.5. Conclusions ..................................................................... 59 2.6. Acknowledgements ......................................................... 60 2.7. References ........................................................................ 60 CHAPTER 3 - ALLELIC DIVERSITY IN THE TRANSCRIPTOMES OF CONTRASTING RUST-INFECTED GENOTYPES OF LATHYRUS SATIVUS, A LASTING RESOURCE FOR SMART BREEDING ...................................... 69 3.1. Abstract ............................................................................ 71 3.2. Introduction ...................................................................... 72 3.3. Material and methods ...................................................... 76 3.3.1. Plant and fungal material, inoculation and RNA isolation ......................................................................... 76 3.3.2. Sequencing and quantification ....................................... 77 3.3.3. SNP detection ................................................................ 79 3.3.4. EST-SSR development and genotyping ......................... 79 3.3.5. Quantitative RT-PCR assay ........................................... 80 3.3.6. Contig annotation and data analysis .............................. 81 3.3.7. Availability of supporting data ........................................ 81 3.4. Results .............................................................................. 82 3.4.1. Contigs from the RNA-Seq transcriptomes of resistant and susceptible L. sativus genotypes ............................ 82 3.4.2. RNA-Seq validation by quantitative RT-PCR assay ....... 83 3.4.3. Differential gene expression in resistant and susceptible L. sativus genotypes during infection ............................. 84 3.4.4. Annotation ..................................................................... 87 3.4.5. Biotic stress related proteins .......................................... 92 vii Table of Contents ___________________________________________________________________ 3.4.6. SNPs in resistance pathways ........................................ 96 3.4.7. EST-SRR development ................................................. 96 3.5. Discussion ........................................................................ 98 3.6. Conclusions ................................................................... 105 3.7. Acknowledgments ......................................................... 106 3.8. References ...................................................................... 106 CHAPTER 4 - LATHYRUS SATIVUS TRANSCRIPTOME RESISTANCE RESPONSE TO ASCOCHYTA LATHYRI INVESTIGATED BY DEEPSUPERSAGE ANALYSIS ........................................................ 115 4.1. Abstract .......................................................................... 117 4.2. Introduction .................................................................... 118 4.3. Material and methods .................................................... 120 4.3.1.1. Plant material and inoculation ...................................... 120 4.3.2. RNA extraction and deepSuperSAGE library construction ................................................................. 120 4.3.3. Data analysis and annotation ....................................... 121 4.3.4. Quantitative RT-PCR assays ....................................... 122 4.3.5. UniTag assignment to functional categories ................ 123 4.4. Results ............................................................................ 125 4.4.1. DeepSuperSAGE library characterization .................... 125 4.4.2. DeepSuperSAGE validation by quantitative RT-PCR assay ........................................................................... 125 4.4.3. Annotation of differentially expressed genes in the resistant L. sativus genotype after A. lathyri infection ....................................................................... 126 4.5. Discussion ...................................................................... 128 4.5.1. Pathogen perception .................................................... 131 viii Table of Contents ___________________________________________________________________ 4.5.2. Hormone signaling ....................................................... 132 4.5.3. Cell wall fortification ..................................................... 134 4.5.4. Antimicrobial activity .................................................... 137 4.5.5. Reactive oxygen species ............................................. 138 4.5.6. Detoxification ............................................................... 138 4.6. Conclusions ................................................................... 139 4.7. Acknowledgments ........................................................
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